Aberrant elevation of FTO levels promotes liver steatosis by decreasing the m6A methylation and increasing the stability of SREBF1 and ChREBP mRNAs

ABSTRACT Previous studies have indicated an association of fat mass and obesity-associated (FTO) with nonalcoholic fatty liver disease (NAFLD), the most common chronic liver disease worldwide. This study aimed to decipher the complex role of FTO in hepatic lipid metabolism. We found that a decrease in N6-methyladenosine (m6A) RNA methylation in the liver of mice fed with a high-fat diet (HFD) was accompanied by an increase in FTO expression. Overexpression of FTO in the liver promoted triglyceride accumulation by upregulating the expression of lipogenic genes. Mechanistical studies revealed that FTO could stabilize the mRNAs of sterol regulatory element binding transcription factor 1 (SREBF1) and carbohydrate responsive element binding protein (ChREBP), two master lipogenic transcription factors, by demethylating m6A sites. Knockdown of either SREBF1 or ChREBP attenuated the lipogenic effect of FTO, suggesting that they are bona fide effectors for FTO in regulating lipogenesis. Insulin could stimulate FTO transcription through a mechanism involving the action of intranuclear insulin receptor beta, while knockdown of FTO abrogated the lipogenic effect of insulin. Inhibition of FTO by entacapone decreased the expression of SREBF1, ChREBP, and downstream lipogenic genes, ameliorating liver steatosis in HFD-fed mice. Thus, our study established a critical role of FTO in both the insulin-regulated hepatic lipogenesis and the pathogenesis of NAFLD and provided a potential strategy for treating NAFLD.


Introduction
Nonalcoholic fatty liver disease ( NAFLD ) has emerged as the most common chronic liver disease, with a global average prevalence ∼25% in adults, higher than type 2 diabetes and obesity ( Powell et al., 2021 ) . It is also the fastest-growing cause of liver-related mortality worldwide, currently with no FDA-approved medication ( Paik et al., 2020 ) . The pathogenesis of NAFLD has been extensively studied; however, the underlying mechanism remains inconclusive. The hallmark of NAFLD is an increase in hepatic triglyceride ( TG ) content. A series of studies suggest that increased TG production rather than decreased mobilization is the primary mechanism for the development of NAFLD ( Sanyal et al., 2001 ;Adiels et al., 2005 ;Bugianesi et al., 2005 ;Fabbrini et al., 2008 ;da Silveira et al., 2020 ) . Many lipogenic enzymes, such as adenosine triphosphate citrate lyase ( ACLY ) , acetyl-CoA carboxylase 1 ( ACC1 ) , fatty acid synthase ( FASN ) , and stearoylcoenzyme A desaturase 1 ( SCD1 ) , are coordinately regulated by key transcription factors, including sterol regulatory element binding protein 1c ( SREBP1c, encoded by SREBF1 ) and carbohydrate responsive element binding protein ( ChREBP ) ( Wang et al., 2015c) . It is noteworthy that the insulin effect on both SREBF1 and ChREBP expression has been noticed before, although the underlying mechanism remains unclear.
FTO has long been associated with obesity according to early genome-wide association studies ( Scuteri et al., 2007 ) and plays a regulatory role in adipogenesis ( Zhao et al., 2014 ;Wang et al., 2015a ) . Mice with the whole-body knockout or lossof-function mutation of FTO had reduced adipose tissue and body weight ( BW ) , while transgenic mice overexpressing FTO displayed increased food intake and obesity ( Fischer et al., 2009 ;Church et al., 2010 ;McMurray et al., 2013 ) . FTO expression increased in the fatty liver of rats and patients with nonalcoholic steatohepatitis ( Guo et al., 2013 ;Lim et al., 2016 ) . It has been suggested that FTO regulates lipogenesis through modulating SREBP1c activity via multiple mechanisms, affecting nuclear translocation, maturation, as well as m 6 A methylation ( Chen et al., 2018 ;Hu et al., 2020 ) . Nevertheless, the physiological role of hepatic FTO during the fasting-feeding cycle and the pathophysiological role of FTO in diet-induced hepatic steatosis in mice remains unclear.
Here, we demonstrated that hepatic FTO could demethylate and stabilize SREBF1 and ChREBP mRNAs, increasing both SREBP1c and ChREBP protein levels and consequently upregulating the expression of key lipogenic enzymes responsible for hepatic lipogenesis. Moreover, insulin could induce the expression of hepatic FTO, which in turn mediated the lipogenic effect of insulin. Furthermore, inhibition of FTO reduced hepatic lipogenesis and TG accumulation, implicating a potential strategy for treating NAFLD.

Reduced m 6 A methylation is accompanied by elevated FTO level in liver steatosis
To investigate whether m 6 A modification is involved in the pathogenesis of hepatic steatosis, we detected the hepatic m 6 A levels in a diet-induced NAFLD mouse model by feeding with high-fat diet ( HFD ) for 4 months. The overall extent of m 6 A methylation in the liver of these mice was substantially reduced ( Figure 1 A ) . Expression analysis of methyltransferases ( writers ) , demethylase ( erasers ) , and methyl binding proteins ( readers ) revealed that both mRNA and protein levels of hepatic FTO and YTHDF2 were increased. Despite an increase of mRNA, the protein level of METTL3 was unchanged ( Figure 1 B and C ) . Considering a decline of overall m 6 A methylation in liver steatosis accompanied by increased FTO expression, we speculated that the demethylase FTO might have a role in hepatic steatosis.

Hepatic FTO overexpression increases TG accumulation in the liver
To understand whether the increased FTO expression contributes to the development of hepatic steatosis, we overexpressed FTO in the liver of chow diet ( CD ) -fed mice by tail vein injection of FTO-expressing adenovirus ( Ad-FTO ) ( Figure 1 D and E; Supplementary Figure S1A ) . Two weeks after injection, mice overexpressing FTO displayed increased liver weight ( LW ) and liver TG content ( Figure 1 F and G ) . Hematoxylin and eosin ( HE ) and Oil-red O staining confirmed that the liver of these mice had histological changes and increased lipid accumulation resembling NAFLD ( Figure 1 H ) . There was no significant increase in the weight of adipose tissues ( Supplementary Figure S1B and C ) . These results suggested that hepatic FTO overexpression induced TG accumulation in the liver under normal CD feeding condition. We also overexpressed FTO in the liver of HFD-fed mice ( Supplementary Figure S1D ) . Three weeks after injection, HFD-fed mice overexpressing FTO exhibited heavier LW, higher liver TG content, and more severe hepatic steatosis as compared with HFD-fed control mice ( Figure 1 I-K ) . No significant change in the weight of adipose tissues was observed ( Supplementary Figure S1E and F ) . These data indicated that hepatic FTO overexpression could aggravate hepatosteatosis under HFD feeding condition. Thus, a high level of FTO might contribute to TG accumulation in the liver.

FTO overexpression promotes hepatic TG accumulation by enhancing lipogenesis
The development of hepatic steatosis is dictated by four aspects: excessive hepatic lipid uptake from systemic and/or portal circulations, increased de novo lipid synthesis in hepatocytes, reduced hepatic lipid oxidation/degradation, and decreased lipid export from hepatocytes ( Suzuki and Diehl, 2017 ) . To decipher which pathway is responsible for hepatosteatosis after FTO overexpression, key genes involved in lipid metabolism were examined. Indeed, mice overexpressing FTO under either normal CD or HFD feeding condition had higher levels of key lipogenic enzymes in the liver, such as ACLY, ACC1, FASN, and SCD1, compared to the respective control mice ( Figure 2 A and B ) . In contrast, no significant change was observed in the mRNA levels of genes involved in fatty acid oxidation ( carnitine palmitoyltransferase I alpha ( CPTI α) and peroxisome proliferator activated receptor alpha ( PPAR α) ) and uptake ( CD36 ) or VLDL exportation ( apolipoprotein B ( APOB ) and microsomal triglyceride transfer protein ( MTTP ) ) (  Figure S2A ) . These results suggested that FTO might promote hepatic TG accumulation primarily by increasing lipogenesis.

Hepatic FTO controls lipogenesis by targeting SREBF1 mRNA
Given that SREBP1c controls the transcription of lipogenic genes ( Wang et al., 2015c) , we tested whether FTO could regulate hepatic lipid metabolism via targeting SREBP1c. We found that both SREBF1 mRNA and SREBP1c protein levels were significantly increased in the liver of mice infected with Ad-FTO under either normal CD or HFD feeding condition, compared to that in control mice under the same feeding condition ( Figure 3 A and B ) . In agreement with the above results, the levels of SREBF1 mRNA and SREBP1c protein were increased in cultured hepatocytes in vitro after Ad-FTO infection ( Figure 3 C and D;Supplementary Figure S2B ) . Moreover, we found that knockdown of FTO by specific small interfering RNA ( siRNA ) could decrease the expression of SREBF1 and its downstream lipogenic genes at both mRNA and protein levels in cultured hepatocytes ( Supplementary Figure  S2C and D ) . These in vivo and in vitro data suggested that FTO might promote hepatic lipogenesis via SREBP1c.
RNA immunoprecipitation ( RIP ) showed enrichment of SREBF1 mRNA in HepG2 cells overexpressing FTO, suggesting direct physical bindings between the FTO protein and SREBF1 mRNA ( Figure 3 E ) . Consistently, RNA methylation immunoprecipitation ( MeRIP ) analysis revealed significantly less enrichment of SREBF1 mRNA in m 6 A-containing transcripts derived from the liver of mice infected with Ad-FTO ( Figure 3 F ) , suggesting that SREBF1 might be a downstream target of FTO. Indeed, m 6 A methylation of SREBF1 mRNA was significantly upregulated after FTO knockdown in HepG2 cells ( Supplementary Figure S2E ) . We identified three previously unrecognized potential m 6 A modification sites in the 3 UTR region of SREBF1 gene ( Supplementary Figure S2F ) . Consistently, a reporter containing these sites showed increased activity after Ad-FTO infection and lower activity after FTO knockdown ( Figure 3 G ) , suggesting that FTO binding on these three m 6 A sites of SREBF1 increased its expression.
As FTO can regulate the stability of the transcripts of their target genes by demethylating m 6 A sites ( Fu et al., 2013 ;Gu et al., 2020 ) , we tested whether FTO expression changes had any effect on the mRNA stability of SREBF1. As expected, the RNA stability assay showed that overexpression of FTO stabilized SREBF1 mRNA in Hepa1-6 cells ( Figure 3 H ) . Collectively, hepatic FTO might promote lipogenesis through enhancing the stability of SREBF1 mRNA via demethylating its m 6 A sites.
To substantiate the pivotal role of SREBP1c in FTO-induced hepatic lipogenesis, we tested whether knockdown of SREBF1 could abolish the effect of FTO ( Figure 3 I and J; Supplementary Figure S2G ) . As expected, SREBF1 knockdown greatly attenuated the effect of FTO overexpression on lipogenic genes, confirming the key role of SREBP1c in mediating the effect of FTO on lipogenesis ( Figure 3 J and K ) . However, knockdown of SREBF1 was not able to totally abolish the effect of FTO overexpression on lipogenic genes ( Figure 3 J and K ) , suggesting that other mechanisms might also be involved.
We also tested whether FTO could regulate the expression of SREBP2, another member of the SREBP family, which is more specific to cholesterogenic gene expression. Ad-FTO infection  Figure S3E and F ) . Furthermore, Ad-FTO treatment did not alter the stability of SREBF2 mRNA ( Supplementary Figure S3G ) . Thus, we concluded that SREBF2 was not under the control of FTO and was not involved in the regulation of hepatic lipid metabolism by FTO.

Hepatic FTO controls lipogenesis by targeting ChREBP mRNA
It has been suggested that hepatic transcription factors besides SREBP1c, such as ChREBP and LXR α, may also contribute to the induction of lipogenic genes. Interestingly, similar to those observed for SREBP1c, both the mRNA and protein levels of ChREBP were significantly elevated in the liver of mice overexpressing FTO under either normal CD or HFD feeding condition, compared to their controls ( Figure Figure S4F ) . Based on these findings, we speculate that SREBP1c and ChREBP, but not LXR α, might be responsible for the regulation of lipogenesis by FTO.
RIP analysis indicated that FTO might demethylate the m 6 A sites of ChREBP mRNA, but not LXR α mRNA, via direct recruitment ( Figure S4H ) . Three potential m 6 A modification sites were identified near the stop codon of ChREBP mRNA ( Supplementary Figure S4K ) . As expected, the activity of the reporter containing these sites increased after Ad-FTO infection and decreased after siFTO transfection ( Figure 4 G ) . In addition, overexpression of FTO stabilized ChREBP mRNA in vitro ( Figure 4 H ) . In contrast, the FTO expression changes had no effect on the m 6 A level and stability of LXR α mRNA ( Supplementary Figure S4I-L ) . Together, these findings indicated that FTO might demethylate and stabilize the mRNA of ChREBP, but not LXR α. As ChREBP is critically involved in the biological effect of glucose, we tested whether the glucose levels in culture medium could affect the regulation of ChREBP by FTO. Given that high-glucose culture medium was normally used in the study, we examined ChREBP expression in HepG2 cells after Ad-FTO infection in low-glucose medium. Under a low-glucose culture condition, FTO overexpression could still increase the mRNA and protein levels of ChREBP ( Supplementary Figure S4M ) , suggesting that the regulation of ChREBP by FTO was independent of glucose.
To clarify the crucial role of ChREBP in FTO-induced hepatic lipogenesis, we tested whether downregulation of ChREBP could attenuate the effect of FTO overexpression in cultured hepatocytes. Notably, ChREBP knockdown blocked the effect of FTO on the expression of key lipogenic genes, suggesting a vital role of ChREBP in mediating the effect of FTO on lipogenesis ( Figure 4 I-K; Supplementary Figure S4N ) . These data suggested that, besides SREBF1, hepatic FTO might also promote lipogenesis through increasing ChREBP by stabilizing its mRNA via m 6 A demethylation.
In addition, we also tested whether SREBP1c and ChREBP could act synergistically as downstream effectors for FTO in the regulation of lipogenic genes. In line with above findings, knockdown of either SREBF1 or ChREBP totally diminished the effect of Ad-FTO on the mRNA levels of lipogenic genes in HepG2 cells, while a cumulative effect was observed after knockdown of both SREBF1 and ChREBP ( Supplementary Figure S5A ) . Similar results were observed for the effect of either SREBF1 or ChREBP knockdown on ACC1 and FASN protein levels after FTO overexpression. However, cumulative effects were not seen after knockdown of both SREBF1 and ChREBP, indicating a compensatory mechanism ( Supplementary Figure S5A ) . On the other hand, overexpression of either SREBF1 or ChREBP could totally offset the effect of FTO knockdown on the mRNA levels of lipogenic genes in HepG2 cells, while a cumulative effect could be observed after overexpression of both SREBF1 and ChREBP ( Supplementary Figure S5B ) . Although similar results could be observed for the effect of either SREBF1 or ChREBP overexpression on ACC1 and FASN protein levels after FTO knockdown, we were not able to detect a cumulative effect of overexpression of both SREBF1 and ChREBP, indicating the presence of a compensatory mechanism ( Supplementary Figure S5B ) . Given that only cumulative but not synergistic effects were observed ( Figures 3 I-K and 4 I-K; Supplementary Figure S5A and B ) , we speculated that either ChREBP or SREBF1 played a critical nonredundant role in the regulation of lipogenic genes by FTO. Thus, instead of concluding that one is more important than the other, both ChREBP and SREBF1 could be bona fide downstream effectors for FTO and their contribution to FTO-controlled lipogenesis might depend

Hepatic FTO is involved in the regulation of lipogenesis by insulin
As the effect of FTO on hepatic lipogenesis resembles that of insulin, a potential link between the insulin signaling pathway and FTO was investigated during the fasting-feeding cycle. After fasting for 6 h, there was a significant increase of m 6 A modification in the liver, accompanied by a decrease of FTO and lipogenic enzymes ( Figure 5 A and B ) . In contrast, re-feeding for 6 h led to the opposite effect ( Figure 5 A and B ) . Consistently, intraperitoneal insulin injection produced similar effects to that of re-feeding in the liver, i.e. a decrease of m 6 A modification and an increase of FTO and lipogenic enzymes ( Recent studies showed that insulin receptor β ( IR β) could translocate into the nucleus and bind to the promoter region of target genes including FTO in cultured hepatocytes after insulin stimulation ( Hancock et al., 2019 ) . Consistently, there was more IR β protein in the nucleus of hepatocytes from insulin-treated mice ( Figure 5 L ) . Moreover, nuclear IR β protein decreased upon fasting and increased upon re-feeding in hepatocytes ( Figure 5 M ) , which was in line with the expression pattern of FTO and the change of m 6 A modification ( Figure 5 A and B ) . There was also more nuclear IR β protein in the hepatocytes of HFD-fed mice ( Figure 5 N ) . These data suggested that IR β might be involved in the regulation of FTO expression by insulin under either physiological or pathological condition.
Additionally, we employed a couple of specific inhibitors for the insulin signaling pathway, targeting different key components downstream of IR β. Specific inhibitors, including LY294002 for PI3K, MK2206 for AKT, and sb415286 for GSK-3, could not reverse the increase of FTO mRNA induced by insulin ( Supplementary Figure S6B and C ) . These results indicated that intranuclear IR β, rather than components of insulin signaling downstream of IR β, played a dominant role in the regulation of FTO expression.

Pharmaceutical FTO inhibition prevents hepatic TG accumulation
Given that the expression of key lipogenic enzymes was elevated in the liver of HFD-fed mice ( Supplementary Figure S7A ) and FTO could positively regulate hepatic lipogenesis, we then employed entacapone ( ENT ) , which is not only a potent inhibitor of FTO but also an FDA-approved drug, to test whether FTO could serve as a potential target to treat hepatosteatosis. Similar to the results observed in FTO knockdown experiments, ENT reduced the expression of both SREBF1 and ChREBP ( but not LXR α) as well as key lipogenic enzymes in hepatocytes ( Figure 6 A-D; Supplementary Figure S7B-D ) . The activity of the reporter containing the SREBF1 or ChREBP fragment with predicted m 6 A sites was reduced after FTO inhibition by ENT treatment ( Figure 6 E ) . Accordingly, RNA stability assay also showed that FTO inhibition by ENT destabilized both SREBF1 and ChREBP ( but not LXR α) mRNAs in hepatocytes ( Figure 6 F and G; Supplementary Figure S7E ) .
To validate the specificity of ENT on FTO inhibition and consequent lipogenic changes, we examined the levels of SREBF1, ChREBP, and lipogenic enzymes after ENT treatment of HepG2 cells overexpressing FTO. ENT treatment greatly attenuated the effect of FTO overexpression on lipogenic genes ( Supplementary Figure S8A ) . Notably, two-way analysis of variance ( ANOVA ) of the quantitative polymerase chain reaction ( qPCR ) results revealed a significant FTO overexpression-by-ENT treatment interaction concerning the mRNA expression of these lipogenic genes, indicating that the ENT treatment-related differences in the mRNA expression of these genes might depend on FTO overexpression ( Supplementary Table S1 ) . On the other hand, the levels of these lipogenic genes after ENT treatment were examined in HepG2 cells with FTO knockdown. The effect of ENT on the expression of these genes was totally abolished after FTO knockdown ( Supplementary Figure S8B ) , further supporting the notion that FTO was required for the ENT action on these lipogenic genes. These conclusions were further substantiated by two-way ANOVA of the qPCR results revealing a significant FTO knockdown-by-ENT treatment interaction concerning the mRNA levels of these lipogenic genes ( Supplementary Table S1 ) . Together, these results suggested that the ENT effect on FTO was specific and the regulatory effect of ENT on the mRNA expression of lipogenic genes was attributed to its inhibitory effect on FTO.
We then treated HFD-fed mice with ENT to examine whether ENT could reduce obesity-associated hepatic TG accumulation. As expected, MeRIP analysis revealed that ENT treatment increased m 6 A modification of SREBF1 and ChREBP mRNAs in the liver of HFD-fed mice ( Figure 6 H ) . The expression of SREBF1 and ChREBP ( but not LXR α and SREBF2 ) and downstream lipogenic genes were all reduced in the liver of HFD-fed mice after ENT treatment (

Discussion
It has been accepted that increased hepatic de novo lipogenesis contributes greatly to the development of steatosis in NAFLD. Although the mechanism responsible for this increase in hepatic lipogenesis is unclear, selective insulin resistance has long been observed in fatty liver, which is manifested by insulin resistance to the suppression of hepatic glucose production but preserves insulin sensitivity in the SREBP1c pathway that stimulates fatty acid synthesis. It has been suggested that hyperinsulinemia activates SREBP1c to promote hepatic TG accumulation in NAFLD patients, though the detailed mechanism remains unclear. FTO is an obesity-susceptible gene identified through genome-wide association studies ( Scuteri et al., 2007 ) . FTO levels were found to be increased in the fatty liver of rodents and patients with NAFLD ( Guo et al., 2013 ;Lim et al., 2016 ) . Although FTO has been implicated in the regulation of hepatic lipogenesis, the physiological or pathophysiological role of FTO in hepatic lipogenesis is not fully understood.
Here, we first observed an increase of FTO in the liver of HFD-fed mice. Then, a causal link between FTO and steatosis was established by in vivo FTO overexpression. Next, we found that FTO induced the expression of genes involved in lipogenesis but not in lipoprotein transport or fatty acid β-oxidation. Following the lead of lipogenesis, we traced back to an increase of its upstream regulators, i.e. SREBP1c and ChREBP, whose mRNAs were direct substrates of FTO. FTO bound to and demethylated m 6 A in SREBF1 and ChREBP mRNAs, enhancing their stability. Since SREBP1c functions in insulin-mediated lipogenesis, we further established that FTO was regulated by insulin and also mediated the lipogenic effect of insulin. Although our data showed that either ChREBP or SREBF1 had a nonredundant role in the regulation of lipogenic genes by FTO, given that only cumulative but not synergistic actions were observed for ChREBP and SREBF1 in hepatocytes in response to FTO, we speculated that these two transcription factors were bona fide downstream effectors for FTO and their contribution to the FTO effect on lipogenesis might depend on physiological and pathological conditions. Here, we deciphered an elegant insulin/FTO/SREBP1c/ChREBP/lipogenesis signaling pathway, consolidating FTO as an important contributor to fatty liver. Through its demethylase activity, FTO added a layer of dynamic gene regulation in hepatic TG metabolism.
A recent study demonstrated that YTH domain-containing 2, an m 6 A reader, could bind to the mRNA of lipogenic genes, decrease their mRNA stability, and inhibit lipogenesis ( Zhou et al., 2021 ) . In light of these findings, m 6 A methylation seems to play an important role in the regulation of hepatic lipid metabolism.
In our study, we observed an increase of FTO in the fatty liver of HFD mice, consistent with previous findings in the fatty liver of rats and patients with nonalcoholic steatohepatitis ( Guo et al., 2013 ;Lim et al., 2016 ) , and a corresponding decrease in m 6 A methylation. We did not find any change in other m 6 A writer or eraser proteins examined in this study. In contrast, a decrease of FTO and an increase of the writer METTL3 has been reported in the liver of db/db mice, accompanied by an increase of global m 6 A methylation ( Zhou et al., 2021 ) . The mechanism underlying this discrepancy is unclear, but it is conceivable that the consequence of m 6 A methylation is complex and context-and genedependent. Results obtained from different models should be interpreted with caution.
As the list of m 6 A target mRNAs is growing, the role of m 6 A modification in cellular, developmental, and disease processes has been attracting extensive attention ( Roundtree et al., 2017 ) . Emerging evidence also suggests the therapeutic potential of targeting m 6 A regulators in diseases. Although FTO has long been linked to obesity, an increasing interest in FTO in metabolic regulation has recently emerged ( Yang et al., 2022 ) . Recent studies from various species or in different metabolic tissues have highlighted the crucial role of FTO in regulating many aspects of lipid homeostasis, including lipogenesis, lipolysis, lipid transportation, lipid uptake, fatty acid oxidation, and adipogenesis, by targeting many genes ( Mo et al., 2017 ;Wu et al., 2017 ;Chen et al., 2018 ;Kang et al., 2018 ;Wei et al., 2022 ) . Nevertheless, relatively few genes, such as CES2, PPAR γ, and PLIN5 ( Mo et al., 2017 ;Takemoto et al., 2021 ;Wei et al., 2021 ) , have been established as direct substrates of FTO. To what extent are these identified targets responsible for the FTO action on lipid metabolism and how they coordinate FTO-controlled lipid metabolism in a context-and tissue-specific manner are not fully understood and require further study in the future.
Structure-based virtual screening identified ENT, an inhibitor of catechol-O-methyltransferase, as a potent FTO chemical inhibitor ( Peng et al., 2019 ) . Previous study in the liver mainly focused on ENT effects on suppressing hepatic gluconeogenesis and improving glucose tolerance. In our study, hepatic FTO inhibition with ENT significantly ameliorated hepatosteatosis in vivo , raising FTO as a promising target for treating diet-induced hepatic metabolic diseases. So far, there is still huge unmet clinical need in the field of NAFLD, without FDA-approved medication available. It would be interesting to develop more FTO inhibitors and test their efficacies in treating NAFLD.
In summary, our study established a functional role of FTO during the fasting-feeding cycle and in the pathogenesis of NAFLD, which responded to insulin and promoted hepatic lipogenesis by stabilizing SREBF1 and ChREBP mRNAs. Our findings provide new insights into the role of m 6 A modification in the regulation of hepatic lipid metabolism in response to hormones  and nutrients. Hepatic FTO targeting strategy deserves to be further explored for its potential in treating NAFLD in the future.

Animal experiments
Male C57BL/6J mice purchased from Shanghai Laboratory Animal Company were kept at 22°C-23°C and 35% ± 5% humidity with a 12-h dark/light cycle, in the specific pathogenfree facility of Shanghai Institute of Nutrition and Health ( SINH ) , Chinese Academy of Sciences ( CAS ) . To induce hepatic steatosis, 4-week-old mice were fed with HFD ( Research Diets, D12492 ) for 16 weeks. Overexpression of FTO in the liver of 8-week-old mice was achieved by tail vein injection of adenoviral FTO ( Ad-FTO ) or control adenovirus ( Ad-Ctrl ) ( 5 × 10 7 pfu/g ) . Two weeks after injection, mice were sacrificed and tissues were collected. HFD-fed mice were also injected with Ad-FTO or Ad-Ctrl ( 5 × 10 7 pfu/g ) . After 3 weeks, mice were sacrificed and tissue samples were collected. For pharmaceutical inhibition of FTO, HFD-fed mice were injected daily with ENT ( 200 mg/kg, CSNpharm, CSN17052 ) or equivalent volume of solvent for 2 weeks. Then, mice were sacrificed, and tissues were collected. All experiments were approved by the Animal Care and Use Committee of SINH, CAS ( 2016-AN-1, SIBS-2019-YH-1 ) .

Liver histology
The fresh liver tissues were fixed in 4% paraformaldehyde for 24 h. Paraffin-embedded samples were sectioned at 5 μm and sequentially stained with HE. For Oil-red O staining, liver tissues were first fixed with sequential 10% and 20% sucrose equilibration, each for 12 h at 4°C, and then cryoembedded in optimal cutting temperature medium. The 10-μm cryostat sections were prepared and stained with Oil-red O for lipids and hematoxylin for nuclei. Finally, HE and Oil-red O staining images were taken using a light microscope ( Olympus ) .

Mouse primary hepatocyte isolation and culture
Mouse primary hepatocyte isolation and culture was performed as previously described ( Liu et al., 2014b ) . Briefly, 8-week-old mice were anaesthetized with sodium pentobarbital ( 30 mg/kg i.p. ) . The portal vein was cannulated under aseptic conditions and the liver was first perfused with phosphatebuffered saline containing 5 mM EGTA and 1% HEPES, and then perfused with digestion buffer containing 0.05% collagenase type I solution. The digestion was quenched by Dulbecco's modified Eagle's medium ( DMEM ) containing 10% fetal bovine serum ( FBS ) . The isolated hepatocytes were filtered through a 70-mm cell strainer, centrifuged, and washed. Finally, hepatocytes were re-suspended with DMEM ( 10% FBS ) and cultured overnight at 1 × 10 6 cells per well in 6-well plates coated with rat-tail collagen type I.

Cell culture and treatment
Hepa1-6 and HepG2 cells were maintained in high-glucose DMEM ( Gibco, C11995500BT ) , unless otherwise indicated, with 10% heat-inactivated FBS. To test whether FTO is involved in the regulation of ChREBP under low-glucose condition, HepG2 cells were cultured in low-glucose DMEM ( Gibco, 31600034 ) with 10% heat-inactivated FBS as indicated. Transfection of siRNA ( 20 nM per well ) was conducted using Lipofectamine RNAiMAX ( Invitrogen, 13778150 ) . The siRNA sequences are listed in Supplementary Table S2. ENT ( 50 μM ) was added when the medium was changed after FTO overexpression or knockdown. To investigate the effect of components of insulin signaling downstream of IR β on FTO mRNA expression, the cells were pretreated with ly294002 ( PI3K inhibitor, 10 μM ) , sb415286 ( GSK-3 inhibitor, 10 μM ) , or mk2206 ( Akt inhibitor, 3 μM ) for 2 h before addition of insulin ( 100 nM ) .

Plasmid construction and adenovirus recombination
The full-length open reading frames ( ORFs ) of human FTO , SREBF1 , and MLXIPL ( ChREBP ) genes were amplified from HepG2 cDNA. The ORF of FTO was cloned into a flag-tagged pcDNA3.1 mammalian expression vector ( Invitrogen, . The ORFs of SREBF1 and MLXIPL were cloned into a pLVX-puro lentiviral expression vector ( Clontech, 632164 ) . m 6 A site prediction was conducted by using a sequence-based m 6 A modification site predictor from the website ( http://www.cuilab.cn/sramp ) . Then, the m 6 A site-containing fragment was amplified from HepG2 cDNA and cloned into the pRL-TK vector for dual-luciferase reporter assay. Recombinant adenovirus for FTO overexpression was generated using the AdEasy Adenoviral Vector System ( Stratagene, RRID:Addgene _ 18703 ) in 293A cells. For in vivo FTO overexpression, viruses were used at 5 × 10 7 pfu/g via tail vein injection. For in vitro transfection using cell lines or primary hepatocytes, viruses were used at the multiplicity of infection of 100. Primers are listed in Supplementary Table S3.

m 6 A RNA methylation quantification
The m 6 A methylation of RNA was quantified by m 6 A RNA methylation Quantification Kit ( colorimetric, ab185912 ) . Briefly, total RNA was bound to the strip wells through RNA high binding solution, followed by adding capture antibody solution and detection antibody solution. The absorbance at 450 nm wavelength was read by a spectrophotometer for colorimetric quantification. The content of m 6 A was directly proportional to the measured optical density value.

mRNA stability assay
Hepa1-6 cells were placed in 12-well plates and cultured overnight. After pretreatment with adenovirus or ENT for 48 h, cells were treated with 5 μg/ml actinomycin D ( act-D, Sigma, a9415 ) to inhibit RNA transcription. Cells were collected at 0, 3, and 6 h after adding act-D. The mRNA expression for each group at the indicated time was calculated and normalized by 18S RNA.

Luciferase assay
HepG2 cells were placed in 48-well plates and cultured overnight. Cells were transfected with adenovirus or siRNA. After 24 h, each well was co-transfected with 0.24 μg PGL3 basic plasmid and 0.02 μg pRL TK plasmid and cultured for another 24 h. Then, the dual luciferase activity was measured by a luminometer with the Dual-Luciferase Reporter Assay System ( Promega, E1960, RRID:SCR _ 020536 ) according to the manufacturer's protocol. The results were represented as the ratio of firefly and Renilla Luciferase activity. m 6 A MeRIP m 6 A MeRIP was measured by using Magna MeRIP m 6 A Kit ( Millipore, 17-10499 ) following the manufacturer's instructions. Briefly, 30 μg RNA was saved as input for qPCR. The m 6 A antibody ( 10 μg ) and magnetic beads ( 50 μl ) were incubated and rotated in IP buffer for 30 min at room temperature. After washing, 300 μg RNA was mixed with beads in IP buffer containing RNase inhibitor, incubated, and rotated overnight at 4°C. Finally, the enriched RNA with m 6 A was eluted, using RNeasy Kit ( Qiagen, 74106 ) . The m 6 A site methylation levels of indicated genes were analyzed by qPCR.

RIP
RIP experiments were performed with the Magna RIP RNA Binding Protein Immunoprecipitation Kit ( Millipore, 17-701 ) . Briefly, flag-tagged FTO ( Flag-FTO ) protein was first expressed by plasmid transfection in HepG2 cells. HepG2 cells were lysed with RIP lysis buffer. Cell lysates were immunoprecipitated with antiflag antibody and incubated with protein A/G magnetic beads overnight at 4°C. The precipitated RNA was purified, reversetranscribed into cDNA, and finally quantified by qPCR.

Data analysis
The experimental data were analyzed and plotted using Excel and Graphpad 7.0 software ( GraphPad Software ) . All data were represented as mean ± standard error of the mean ( SEM ) of at least three independent experiments. Statistical significance was assessed by an unpaired Student's t-test unless otherwise indicated. To evaluate the interaction between two independent variables, two-way ANOVA analysis with Tukey's multiple comparison test was used as indicated. A P -value of < 0.05 was considered to be of statistical significance.

Supplementary material
Supplementary material is available at Journal of Molecular Cell Biology online.